mant gdp  (Jena Bioscience)


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  • 94
    Name:
    Mant GDP
    Description:

    Catalog Number:
    nu-204l
    Price:
    343.57
    Applications:
    Conformational dynamic: DnaB/C-proteins[1], Csk[2] Fluorescence stop-flow kinetics: release factor[3], elF5B[4], EF-1B[5], EF-Tu[6] FRET: AC[7]
    Purity:
    ≥ 95 % (HPLC)
    Category:
    Nucleotides Nucleosides
    Buy from Supplier


    Structured Review

    Jena Bioscience mant gdp
    Replacement of <t>mant-GMPPNP</t> by <t>mant-GDP</t> eliminates the dissociation of Rac1 · RhoGDI complexes by anionic liposomes and TrioN, but nucleotide exchange on Rac1 in complex with RhoGDI is conserved. 2 nmol of Rac1(GDP)·RhoGDI complex and 10

    https://www.bioz.com/result/mant gdp/product/Jena Bioscience
    Average 94 stars, based on 33 article reviews
    Price from $9.99 to $1999.99
    mant gdp - by Bioz Stars, 2020-08
    94/100 stars

    Images

    1) Product Images from "Dissociation of Rac1(GDP)?RhoGDI Complexes by the Cooperative Action of Anionic Liposomes Containing Phosphatidylinositol 3,4,5-Trisphosphate, Rac Guanine Nucleotide Exchange Factor, and GTP *"

    Article Title: Dissociation of Rac1(GDP)?RhoGDI Complexes by the Cooperative Action of Anionic Liposomes Containing Phosphatidylinositol 3,4,5-Trisphosphate, Rac Guanine Nucleotide Exchange Factor, and GTP *

    Journal:

    doi: 10.1074/jbc.M800734200

    Replacement of mant-GMPPNP by mant-GDP eliminates the dissociation of Rac1 · RhoGDI complexes by anionic liposomes and TrioN, but nucleotide exchange on Rac1 in complex with RhoGDI is conserved. 2 nmol of Rac1(GDP)·RhoGDI complex and 10
    Figure Legend Snippet: Replacement of mant-GMPPNP by mant-GDP eliminates the dissociation of Rac1 · RhoGDI complexes by anionic liposomes and TrioN, but nucleotide exchange on Rac1 in complex with RhoGDI is conserved. 2 nmol of Rac1(GDP)·RhoGDI complex and 10

    Techniques Used:

    Exposure of Rac1(GDP) · RhoGDI complexes to anionic liposomes, mant-GMMPNP, and TrioN causes dissociation of complexes, GDP to mant-GMPPNP exchange on Rac1, and binding of Rac1-GMPPNP to liposomes. 2 nmol of Rac1(GDP)·RhoGDI complex were
    Figure Legend Snippet: Exposure of Rac1(GDP) · RhoGDI complexes to anionic liposomes, mant-GMMPNP, and TrioN causes dissociation of complexes, GDP to mant-GMPPNP exchange on Rac1, and binding of Rac1-GMPPNP to liposomes. 2 nmol of Rac1(GDP)·RhoGDI complex were

    Techniques Used: Binding Assay

    2) Product Images from "The GTPase Activity of Murine Guanylate-binding Protein 2 (mGBP2) Controls the Intracellular Localization and Recruitment to the Parasitophorous Vacuole of Toxoplasma gondii *"

    Article Title: The GTPase Activity of Murine Guanylate-binding Protein 2 (mGBP2) Controls the Intracellular Localization and Recruitment to the Parasitophorous Vacuole of Toxoplasma gondii *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M112.379636

    Nucleotide-dependent multimerization. Size-exclusion chromatography of WT ( A ) and mutant mGBP2 R48A ( B ), K51A ( C ), E99A ( D ), and D182N ( E ) bound to GTPγS, GDP, GMP, and in the nucleotide-free state at 4 °C. Elution of all proteins was
    Figure Legend Snippet: Nucleotide-dependent multimerization. Size-exclusion chromatography of WT ( A ) and mutant mGBP2 R48A ( B ), K51A ( C ), E99A ( D ), and D182N ( E ) bound to GTPγS, GDP, GMP, and in the nucleotide-free state at 4 °C. Elution of all proteins was

    Techniques Used: Size-exclusion Chromatography, Mutagenesis

    3) Product Images from "Circularly permuted GTPase YqeH binds 30S ribosomal subunit: Implications for its role in ribosome assembly"

    Article Title: Circularly permuted GTPase YqeH binds 30S ribosomal subunit: Implications for its role in ribosome assembly

    Journal: Biochemical and Biophysical Research Communications

    doi: 10.1016/j.bbrc.2009.06.078

    YqeH binds 30S ribosomal subunit in the presence of GDPNP/GTP. (A) Co-sedimentation experiments were carried out by incubating purified ribosomes with GST-YqeH (or GST-YlqF) and nucleotides. Following centrifugation, the absorbance at 254 nm was monitored for the fractions collected (see Supplementary material for details). A representative ribosome profile thus obtained is shown here and peaks corresponding to 30S and 50S subunits are marked. Peaks corresponding to fractions 6 and 9 contain 30S and 50S subunits (see Supplementary material: Fig. Sl ). (b) Emission spectra (400–550 nm) of fluorescent mant-GDP and mant-GDPNP excited at 355 nm are shown with and without YqeH, as displayed in the inset. The fluorescence intensity is shown in arbitrary units (a.u.). (C–K) Fractions collected following co-sedimentation experiments with GST, GST-YlqF and GST-YqeH or its derivatives, were probed using anti-GST antibody in a Western blot. Shown on the right side of the gels (C–K) are the proteins used, indicating the domains they possess. (C) Purified GST was used as a negative control, as all constructs carry an N-terminal GST tag. (D) YlqF, which interacts with 50S, was used as a marker to identify the 50S fractions and as a positive control. Deletion constructs (H) ΔC-YqeH (residues 1–224) and (I) ΔN-YqeH (residues 64–366) and the stand-alone Zn finger (residues 1–46) domain (J) and PNR (residues 225–366) domain (K) are also indicated. The fractions corresponding to the ribosome profile are shown on the top and the nucleotides (GDP/GTP/GDPNP) used are indicated on the left. A high stoichiometric ratio of nucleotide (1 mM) to protein (500 nM) was used to ensure the desired nucleotide bound state.
    Figure Legend Snippet: YqeH binds 30S ribosomal subunit in the presence of GDPNP/GTP. (A) Co-sedimentation experiments were carried out by incubating purified ribosomes with GST-YqeH (or GST-YlqF) and nucleotides. Following centrifugation, the absorbance at 254 nm was monitored for the fractions collected (see Supplementary material for details). A representative ribosome profile thus obtained is shown here and peaks corresponding to 30S and 50S subunits are marked. Peaks corresponding to fractions 6 and 9 contain 30S and 50S subunits (see Supplementary material: Fig. Sl ). (b) Emission spectra (400–550 nm) of fluorescent mant-GDP and mant-GDPNP excited at 355 nm are shown with and without YqeH, as displayed in the inset. The fluorescence intensity is shown in arbitrary units (a.u.). (C–K) Fractions collected following co-sedimentation experiments with GST, GST-YlqF and GST-YqeH or its derivatives, were probed using anti-GST antibody in a Western blot. Shown on the right side of the gels (C–K) are the proteins used, indicating the domains they possess. (C) Purified GST was used as a negative control, as all constructs carry an N-terminal GST tag. (D) YlqF, which interacts with 50S, was used as a marker to identify the 50S fractions and as a positive control. Deletion constructs (H) ΔC-YqeH (residues 1–224) and (I) ΔN-YqeH (residues 64–366) and the stand-alone Zn finger (residues 1–46) domain (J) and PNR (residues 225–366) domain (K) are also indicated. The fractions corresponding to the ribosome profile are shown on the top and the nucleotides (GDP/GTP/GDPNP) used are indicated on the left. A high stoichiometric ratio of nucleotide (1 mM) to protein (500 nM) was used to ensure the desired nucleotide bound state.

    Techniques Used: Sedimentation, Purification, Centrifugation, Fluorescence, Western Blot, Negative Control, Construct, Marker, Positive Control

    4) Product Images from "Circularly permuted GTPase YqeH binds 30S ribosomal subunit: Implications for its role in ribosome assembly"

    Article Title: Circularly permuted GTPase YqeH binds 30S ribosomal subunit: Implications for its role in ribosome assembly

    Journal: Biochemical and Biophysical Research Communications

    doi: 10.1016/j.bbrc.2009.06.078

    YqeH binds a non-specific RNA. EMSA was carried out with YqeH or the indicated domains, in presence of either ssRNA or dsRNA. The migration of ssRNA and dsRNA is shown as control in the lanes at the extreme left and is indicated by an arrow. A retarded migration of RNA in presence of YqeH is indicated by ’shift’. The nucleotide bound states of YqeH are indicated on the top. (A) Both ssRNA and dsRNA are retarded in the presence of YqeH. The dissociation of dsRNA into ssRNAs is not observed in nucleotide-free, GDP and GTP-bound forms. The presence of S5 is indicated above the lanes. The apparent reduction in the intensity of free dsRNAs in the presence of S5 when compared to the corresponding lanes containing YqeH alone suggests a potential increase in YqeH–RNA interactions (the last three lanes in the right). (B) Like in (A), the migration of ssRNA and the mixture of complementary ssRNAs is shown in the lanes at the extreme left as controls and is indicated by an arrow. Increasing concentration of YqeH (2.5, 5, 10 μM) is depicted by a triangle on the top. No annealing activity for YqeH was apparent. (C) EMSA carried out with GST, N (NTD) and C (CTD) terminal domains of YqeH (indicated above the lanes) showed no apparent shift in migration of ssRNA and dsRNA. (D) Also in the presence of deletion constructs (ΔN and ΔC-YqeH), no shift in dsRNA migration was observed.
    Figure Legend Snippet: YqeH binds a non-specific RNA. EMSA was carried out with YqeH or the indicated domains, in presence of either ssRNA or dsRNA. The migration of ssRNA and dsRNA is shown as control in the lanes at the extreme left and is indicated by an arrow. A retarded migration of RNA in presence of YqeH is indicated by ’shift’. The nucleotide bound states of YqeH are indicated on the top. (A) Both ssRNA and dsRNA are retarded in the presence of YqeH. The dissociation of dsRNA into ssRNAs is not observed in nucleotide-free, GDP and GTP-bound forms. The presence of S5 is indicated above the lanes. The apparent reduction in the intensity of free dsRNAs in the presence of S5 when compared to the corresponding lanes containing YqeH alone suggests a potential increase in YqeH–RNA interactions (the last three lanes in the right). (B) Like in (A), the migration of ssRNA and the mixture of complementary ssRNAs is shown in the lanes at the extreme left as controls and is indicated by an arrow. Increasing concentration of YqeH (2.5, 5, 10 μM) is depicted by a triangle on the top. No annealing activity for YqeH was apparent. (C) EMSA carried out with GST, N (NTD) and C (CTD) terminal domains of YqeH (indicated above the lanes) showed no apparent shift in migration of ssRNA and dsRNA. (D) Also in the presence of deletion constructs (ΔN and ΔC-YqeH), no shift in dsRNA migration was observed.

    Techniques Used: Migration, Concentration Assay, Activity Assay, Construct

    YqeH binds 30S ribosomal subunit in the presence of GDPNP/GTP. (A) Co-sedimentation experiments were carried out by incubating purified ribosomes with GST-YqeH (or GST-YlqF) and nucleotides. Following centrifugation, the absorbance at 254 nm was monitored for the fractions collected (see Supplementary material for details). A representative ribosome profile thus obtained is shown here and peaks corresponding to 30S and 50S subunits are marked. Peaks corresponding to fractions 6 and 9 contain 30S and 50S subunits (see Supplementary material: Fig. Sl ). (b) Emission spectra (400–550 nm) of fluorescent mant-GDP and mant-GDPNP excited at 355 nm are shown with and without YqeH, as displayed in the inset. The fluorescence intensity is shown in arbitrary units (a.u.). (C–K) Fractions collected following co-sedimentation experiments with GST, GST-YlqF and GST-YqeH or its derivatives, were probed using anti-GST antibody in a Western blot. Shown on the right side of the gels (C–K) are the proteins used, indicating the domains they possess. (C) Purified GST was used as a negative control, as all constructs carry an N-terminal GST tag. (D) YlqF, which interacts with 50S, was used as a marker to identify the 50S fractions and as a positive control. Deletion constructs (H) ΔC-YqeH (residues 1–224) and (I) ΔN-YqeH (residues 64–366) and the stand-alone Zn finger (residues 1–46) domain (J) and PNR (residues 225–366) domain (K) are also indicated. The fractions corresponding to the ribosome profile are shown on the top and the nucleotides (GDP/GTP/GDPNP) used are indicated on the left. A high stoichiometric ratio of nucleotide (1 mM) to protein (500 nM) was used to ensure the desired nucleotide bound state.
    Figure Legend Snippet: YqeH binds 30S ribosomal subunit in the presence of GDPNP/GTP. (A) Co-sedimentation experiments were carried out by incubating purified ribosomes with GST-YqeH (or GST-YlqF) and nucleotides. Following centrifugation, the absorbance at 254 nm was monitored for the fractions collected (see Supplementary material for details). A representative ribosome profile thus obtained is shown here and peaks corresponding to 30S and 50S subunits are marked. Peaks corresponding to fractions 6 and 9 contain 30S and 50S subunits (see Supplementary material: Fig. Sl ). (b) Emission spectra (400–550 nm) of fluorescent mant-GDP and mant-GDPNP excited at 355 nm are shown with and without YqeH, as displayed in the inset. The fluorescence intensity is shown in arbitrary units (a.u.). (C–K) Fractions collected following co-sedimentation experiments with GST, GST-YlqF and GST-YqeH or its derivatives, were probed using anti-GST antibody in a Western blot. Shown on the right side of the gels (C–K) are the proteins used, indicating the domains they possess. (C) Purified GST was used as a negative control, as all constructs carry an N-terminal GST tag. (D) YlqF, which interacts with 50S, was used as a marker to identify the 50S fractions and as a positive control. Deletion constructs (H) ΔC-YqeH (residues 1–224) and (I) ΔN-YqeH (residues 64–366) and the stand-alone Zn finger (residues 1–46) domain (J) and PNR (residues 225–366) domain (K) are also indicated. The fractions corresponding to the ribosome profile are shown on the top and the nucleotides (GDP/GTP/GDPNP) used are indicated on the left. A high stoichiometric ratio of nucleotide (1 mM) to protein (500 nM) was used to ensure the desired nucleotide bound state.

    Techniques Used: Sedimentation, Purification, Centrifugation, Fluorescence, Western Blot, Negative Control, Construct, Marker, Positive Control

    5) Product Images from "The GTPase Activity of Murine Guanylate-binding Protein 2 (mGBP2) Controls the Intracellular Localization and Recruitment to the Parasitophorous Vacuole of Toxoplasma gondii *"

    Article Title: The GTPase Activity of Murine Guanylate-binding Protein 2 (mGBP2) Controls the Intracellular Localization and Recruitment to the Parasitophorous Vacuole of Toxoplasma gondii *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M112.379636

    Nucleotide-dependent multimerization. Size-exclusion chromatography of WT ( A ) and mutant mGBP2 R48A ( B ), K51A ( C ), E99A ( D ), and D182N ( E ) bound to GTPγS, GDP, GMP, and in the nucleotide-free state at 4 °C. Elution of all proteins was
    Figure Legend Snippet: Nucleotide-dependent multimerization. Size-exclusion chromatography of WT ( A ) and mutant mGBP2 R48A ( B ), K51A ( C ), E99A ( D ), and D182N ( E ) bound to GTPγS, GDP, GMP, and in the nucleotide-free state at 4 °C. Elution of all proteins was

    Techniques Used: Size-exclusion Chromatography, Mutagenesis

    6) Product Images from "Role and timing of GTP binding and hydrolysis during EF-G-dependent tRNA translocation on the ribosome"

    Article Title: Role and timing of GTP binding and hydrolysis during EF-G-dependent tRNA translocation on the ribosome

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    doi: 10.1073/pnas.0606099103

    GTP/GDP binding to EF-G. ( A ) Fluorescence titrations with mant-GTP (circles) and mant-GDP (triangles). ( B ) TLC of mant-GTP and mant-GDP. ( C ) Chase titrations with GTP (circles) or GDP (triangles) monitoring the fluorescence of Bodipy FL-GDP. ( D ) Titrations monitoring tryptophan fluorescence. GTP (circles) or GDP (triangles). ( Inset ) Concentration dependence of GTP binding kinetics. k on = 0.58 ± 0.04 μM −1 ·s −1 ; k off = 13 ± 1 s −1 . ( E ) Binding of [ 3 H]GDP (triangles) or [ 3 H]GTP (circles) to nitrocellulose filters in the presence (filled symbols) or absence (open symbols) of EF-G. ( F ) Retention of EF-G·[ 3 H]GDP (triangles) and EF-G·[ 3 H]GTP (circles) on nitrocellulose filters. Shown are difference titration curves from E . In A – D , continuous lines represent fits (see Materials and Methods ).
    Figure Legend Snippet: GTP/GDP binding to EF-G. ( A ) Fluorescence titrations with mant-GTP (circles) and mant-GDP (triangles). ( B ) TLC of mant-GTP and mant-GDP. ( C ) Chase titrations with GTP (circles) or GDP (triangles) monitoring the fluorescence of Bodipy FL-GDP. ( D ) Titrations monitoring tryptophan fluorescence. GTP (circles) or GDP (triangles). ( Inset ) Concentration dependence of GTP binding kinetics. k on = 0.58 ± 0.04 μM −1 ·s −1 ; k off = 13 ± 1 s −1 . ( E ) Binding of [ 3 H]GDP (triangles) or [ 3 H]GTP (circles) to nitrocellulose filters in the presence (filled symbols) or absence (open symbols) of EF-G. ( F ) Retention of EF-G·[ 3 H]GDP (triangles) and EF-G·[ 3 H]GTP (circles) on nitrocellulose filters. Shown are difference titration curves from E . In A – D , continuous lines represent fits (see Materials and Methods ).

    Techniques Used: Binding Assay, Fluorescence, Thin Layer Chromatography, Concentration Assay, Titration

    Translocation with GTP, GDP, and GDPNP. Time courses of translocation were measured by fluorescence stopped-flow, monitoring the fluorescence of proflavin-labeled fMetPhe-tRNA Phe (traces 1, 4, and 6) or of mRNA(fluorescein + 14) (traces 2, 3, and 5). Time courses were evaluated by single-exponential fitting (see Materials and Methods ) to obtain the following values for k app (SD ±15%): trace 1, GTP, 21 s −1 ; trace 2, GTP, 18 s −1 ; trace 3, GDPNP, 0.8 s −1 ; traces 4 and 5, GDP, 0.9 s −1 ; trace 6, mant-GDP, 0.6 s −1 .
    Figure Legend Snippet: Translocation with GTP, GDP, and GDPNP. Time courses of translocation were measured by fluorescence stopped-flow, monitoring the fluorescence of proflavin-labeled fMetPhe-tRNA Phe (traces 1, 4, and 6) or of mRNA(fluorescein + 14) (traces 2, 3, and 5). Time courses were evaluated by single-exponential fitting (see Materials and Methods ) to obtain the following values for k app (SD ±15%): trace 1, GTP, 21 s −1 ; trace 2, GTP, 18 s −1 ; trace 3, GDPNP, 0.8 s −1 ; traces 4 and 5, GDP, 0.9 s −1 ; trace 6, mant-GDP, 0.6 s −1 .

    Techniques Used: Translocation Assay, Fluorescence, Flow Cytometry, Labeling

    7) Product Images from "Circularly permuted GTPase YqeH binds 30S ribosomal subunit: Implications for its role in ribosome assembly"

    Article Title: Circularly permuted GTPase YqeH binds 30S ribosomal subunit: Implications for its role in ribosome assembly

    Journal: Biochemical and Biophysical Research Communications

    doi: 10.1016/j.bbrc.2009.06.078

    YqeH binds a non-specific RNA. EMSA was carried out with YqeH or the indicated domains, in presence of either ssRNA or dsRNA. The migration of ssRNA and dsRNA is shown as control in the lanes at the extreme left and is indicated by an arrow. A retarded migration of RNA in presence of YqeH is indicated by ’shift’. The nucleotide bound states of YqeH are indicated on the top. (A) Both ssRNA and dsRNA are retarded in the presence of YqeH. The dissociation of dsRNA into ssRNAs is not observed in nucleotide-free, GDP and GTP-bound forms. The presence of S5 is indicated above the lanes. The apparent reduction in the intensity of free dsRNAs in the presence of S5 when compared to the corresponding lanes containing YqeH alone suggests a potential increase in YqeH–RNA interactions (the last three lanes in the right). (B) Like in (A), the migration of ssRNA and the mixture of complementary ssRNAs is shown in the lanes at the extreme left as controls and is indicated by an arrow. Increasing concentration of YqeH (2.5, 5, 10 μM) is depicted by a triangle on the top. No annealing activity for YqeH was apparent. (C) EMSA carried out with GST, N (NTD) and C (CTD) terminal domains of YqeH (indicated above the lanes) showed no apparent shift in migration of ssRNA and dsRNA. (D) Also in the presence of deletion constructs (ΔN and ΔC-YqeH), no shift in dsRNA migration was observed.
    Figure Legend Snippet: YqeH binds a non-specific RNA. EMSA was carried out with YqeH or the indicated domains, in presence of either ssRNA or dsRNA. The migration of ssRNA and dsRNA is shown as control in the lanes at the extreme left and is indicated by an arrow. A retarded migration of RNA in presence of YqeH is indicated by ’shift’. The nucleotide bound states of YqeH are indicated on the top. (A) Both ssRNA and dsRNA are retarded in the presence of YqeH. The dissociation of dsRNA into ssRNAs is not observed in nucleotide-free, GDP and GTP-bound forms. The presence of S5 is indicated above the lanes. The apparent reduction in the intensity of free dsRNAs in the presence of S5 when compared to the corresponding lanes containing YqeH alone suggests a potential increase in YqeH–RNA interactions (the last three lanes in the right). (B) Like in (A), the migration of ssRNA and the mixture of complementary ssRNAs is shown in the lanes at the extreme left as controls and is indicated by an arrow. Increasing concentration of YqeH (2.5, 5, 10 μM) is depicted by a triangle on the top. No annealing activity for YqeH was apparent. (C) EMSA carried out with GST, N (NTD) and C (CTD) terminal domains of YqeH (indicated above the lanes) showed no apparent shift in migration of ssRNA and dsRNA. (D) Also in the presence of deletion constructs (ΔN and ΔC-YqeH), no shift in dsRNA migration was observed.

    Techniques Used: Migration, Concentration Assay, Activity Assay, Construct

    YqeH binds 30S ribosomal subunit in the presence of GDPNP/GTP. (A) Co-sedimentation experiments were carried out by incubating purified ribosomes with GST-YqeH (or GST-YlqF) and nucleotides. Following centrifugation, the absorbance at 254 nm was monitored for the fractions collected (see Supplementary material for details). A representative ribosome profile thus obtained is shown here and peaks corresponding to 30S and 50S subunits are marked. Peaks corresponding to fractions 6 and 9 contain 30S and 50S subunits (see Supplementary material: Fig. Sl ). (b) Emission spectra (400–550 nm) of fluorescent mant-GDP and mant-GDPNP excited at 355 nm are shown with and without YqeH, as displayed in the inset. The fluorescence intensity is shown in arbitrary units (a.u.). (C–K) Fractions collected following co-sedimentation experiments with GST, GST-YlqF and GST-YqeH or its derivatives, were probed using anti-GST antibody in a Western blot. Shown on the right side of the gels (C–K) are the proteins used, indicating the domains they possess. (C) Purified GST was used as a negative control, as all constructs carry an N-terminal GST tag. (D) YlqF, which interacts with 50S, was used as a marker to identify the 50S fractions and as a positive control. Deletion constructs (H) ΔC-YqeH (residues 1–224) and (I) ΔN-YqeH (residues 64–366) and the stand-alone Zn finger (residues 1–46) domain (J) and PNR (residues 225–366) domain (K) are also indicated. The fractions corresponding to the ribosome profile are shown on the top and the nucleotides (GDP/GTP/GDPNP) used are indicated on the left. A high stoichiometric ratio of nucleotide (1 mM) to protein (500 nM) was used to ensure the desired nucleotide bound state.
    Figure Legend Snippet: YqeH binds 30S ribosomal subunit in the presence of GDPNP/GTP. (A) Co-sedimentation experiments were carried out by incubating purified ribosomes with GST-YqeH (or GST-YlqF) and nucleotides. Following centrifugation, the absorbance at 254 nm was monitored for the fractions collected (see Supplementary material for details). A representative ribosome profile thus obtained is shown here and peaks corresponding to 30S and 50S subunits are marked. Peaks corresponding to fractions 6 and 9 contain 30S and 50S subunits (see Supplementary material: Fig. Sl ). (b) Emission spectra (400–550 nm) of fluorescent mant-GDP and mant-GDPNP excited at 355 nm are shown with and without YqeH, as displayed in the inset. The fluorescence intensity is shown in arbitrary units (a.u.). (C–K) Fractions collected following co-sedimentation experiments with GST, GST-YlqF and GST-YqeH or its derivatives, were probed using anti-GST antibody in a Western blot. Shown on the right side of the gels (C–K) are the proteins used, indicating the domains they possess. (C) Purified GST was used as a negative control, as all constructs carry an N-terminal GST tag. (D) YlqF, which interacts with 50S, was used as a marker to identify the 50S fractions and as a positive control. Deletion constructs (H) ΔC-YqeH (residues 1–224) and (I) ΔN-YqeH (residues 64–366) and the stand-alone Zn finger (residues 1–46) domain (J) and PNR (residues 225–366) domain (K) are also indicated. The fractions corresponding to the ribosome profile are shown on the top and the nucleotides (GDP/GTP/GDPNP) used are indicated on the left. A high stoichiometric ratio of nucleotide (1 mM) to protein (500 nM) was used to ensure the desired nucleotide bound state.

    Techniques Used: Sedimentation, Purification, Centrifugation, Fluorescence, Western Blot, Negative Control, Construct, Marker, Positive Control

    8) Product Images from "The GTPase Activity of Murine Guanylate-binding Protein 2 (mGBP2) Controls the Intracellular Localization and Recruitment to the Parasitophorous Vacuole of Toxoplasma gondii *"

    Article Title: The GTPase Activity of Murine Guanylate-binding Protein 2 (mGBP2) Controls the Intracellular Localization and Recruitment to the Parasitophorous Vacuole of Toxoplasma gondii *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M112.379636

    Establishment of the transition state. Size-exclusion chromatography of the WT protein, K51A, and R48A mGBP2 mutants in the GDP·AlF x -bound form. Elution of all proteins was followed using absorbance by 280 nm. The protein size was estimated by
    Figure Legend Snippet: Establishment of the transition state. Size-exclusion chromatography of the WT protein, K51A, and R48A mGBP2 mutants in the GDP·AlF x -bound form. Elution of all proteins was followed using absorbance by 280 nm. The protein size was estimated by

    Techniques Used: Size-exclusion Chromatography

    Nucleotide-dependent multimerization. Size-exclusion chromatography of WT ( A ) and mutant mGBP2 R48A ( B ), K51A ( C ), E99A ( D ), and D182N ( E ) bound to GTPγS, GDP, GMP, and in the nucleotide-free state at 4 °C. Elution of all proteins was
    Figure Legend Snippet: Nucleotide-dependent multimerization. Size-exclusion chromatography of WT ( A ) and mutant mGBP2 R48A ( B ), K51A ( C ), E99A ( D ), and D182N ( E ) bound to GTPγS, GDP, GMP, and in the nucleotide-free state at 4 °C. Elution of all proteins was

    Techniques Used: Size-exclusion Chromatography, Mutagenesis

    9) Product Images from "The GTPase Activity of Murine Guanylate-binding Protein 2 (mGBP2) Controls the Intracellular Localization and Recruitment to the Parasitophorous Vacuole of Toxoplasma gondii *"

    Article Title: The GTPase Activity of Murine Guanylate-binding Protein 2 (mGBP2) Controls the Intracellular Localization and Recruitment to the Parasitophorous Vacuole of Toxoplasma gondii *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M112.379636

    Nucleotide-dependent multimerization. Size-exclusion chromatography of WT ( A ) and mutant mGBP2 R48A ( B ), K51A ( C ), E99A ( D ), and D182N ( E ) bound to GTPγS, GDP, GMP, and in the nucleotide-free state at 4 °C. Elution of all proteins was
    Figure Legend Snippet: Nucleotide-dependent multimerization. Size-exclusion chromatography of WT ( A ) and mutant mGBP2 R48A ( B ), K51A ( C ), E99A ( D ), and D182N ( E ) bound to GTPγS, GDP, GMP, and in the nucleotide-free state at 4 °C. Elution of all proteins was

    Techniques Used: Size-exclusion Chromatography, Mutagenesis

    10) Product Images from "The activation mechanism of Irga6, an interferon-inducible GTPase contributing to mouse resistance against Toxoplasma gondii"

    Article Title: The activation mechanism of Irga6, an interferon-inducible GTPase contributing to mouse resistance against Toxoplasma gondii

    Journal: BMC Biology

    doi: 10.1186/1741-7007-9-7

    The Glu106 is essential for the activation of GTP hydrolysis . (a and b) View of the nucleotide-binding region. (a) The Irga6 dimer model (Figure 4) is shown (cyan and magenta). The cis interaction between the Glu106 and the γ-phosphate, and the putative trans interactions between the 3'OH and Glu106, as well as between the 3'OH and the γ-phosphate are represented by dotted lines. (b) Two molecules (cyan and magenta) of Irga6 bound to GDP (PDB 1TPZ/A ) [ 14 ] were adjusted to the Irga6 dimer model, to give the best overlay for the G1, G3, G4 and G5-motifs. The resulting theoretical model of the
    Figure Legend Snippet: The Glu106 is essential for the activation of GTP hydrolysis . (a and b) View of the nucleotide-binding region. (a) The Irga6 dimer model (Figure 4) is shown (cyan and magenta). The cis interaction between the Glu106 and the γ-phosphate, and the putative trans interactions between the 3'OH and Glu106, as well as between the 3'OH and the γ-phosphate are represented by dotted lines. (b) Two molecules (cyan and magenta) of Irga6 bound to GDP (PDB 1TPZ/A ) [ 14 ] were adjusted to the Irga6 dimer model, to give the best overlay for the G1, G3, G4 and G5-motifs. The resulting theoretical model of the "Irga6 dimer in the GDP state" is shown. (c) Oligomerisation of 80 μM WT or mutant Irga6 proteins was monitored by light scattering in the presence of 10 mM GTP at 37°C. (d) Hydrolysis of 10 mM GTP (with traces α 32 P-GTP) was measured in the presence of 80 μM WT or mutant Irga6 proteins at 37°C. Samples were assayed by TLC and autoradiography.

    Techniques Used: Activation Assay, Binding Assay, Mutagenesis, Thin Layer Chromatography, Autoradiography

    The catalytic interface is involved in Irga6-Irgb6 and Irga6-Irgm3 interactions . Pull-down of Irgb6 (a) and Irgm3 (b) with recombinant GST-tagged Irga6 protein from IFNγ-stimulated gs3T3 fibroblasts lysate in the presence or absence of guanine nucleotides (0.5 mM GDP, GTPγS or mant-GDP). GST-Irga6 protein was visualised by Ponceau S staining upon blotting (top rows). Irgb6 and Irgm3 were detected with anti-Irgb6 and anti-Irgm3 monoclonal antibodies (bottom rows). A shorter exposure of the lysate input is shown. Dotted lines indicate positions where irrelevant lanes were removed from the image.
    Figure Legend Snippet: The catalytic interface is involved in Irga6-Irgb6 and Irga6-Irgm3 interactions . Pull-down of Irgb6 (a) and Irgm3 (b) with recombinant GST-tagged Irga6 protein from IFNγ-stimulated gs3T3 fibroblasts lysate in the presence or absence of guanine nucleotides (0.5 mM GDP, GTPγS or mant-GDP). GST-Irga6 protein was visualised by Ponceau S staining upon blotting (top rows). Irgb6 and Irgm3 were detected with anti-Irgb6 and anti-Irgm3 monoclonal antibodies (bottom rows). A shorter exposure of the lysate input is shown. Dotted lines indicate positions where irrelevant lanes were removed from the image.

    Techniques Used: Recombinant, Staining

    11) Product Images from "Circularly permuted GTPase YqeH binds 30S ribosomal subunit: Implications for its role in ribosome assembly"

    Article Title: Circularly permuted GTPase YqeH binds 30S ribosomal subunit: Implications for its role in ribosome assembly

    Journal: Biochemical and Biophysical Research Communications

    doi: 10.1016/j.bbrc.2009.06.078

    YqeH binds 30S ribosomal subunit in the presence of GDPNP/GTP. (A) Co-sedimentation experiments were carried out by incubating purified ribosomes with GST-YqeH (or GST-YlqF) and nucleotides. Following centrifugation, the absorbance at 254 nm was monitored for the fractions collected (see Supplementary material for details). A representative ribosome profile thus obtained is shown here and peaks corresponding to 30S and 50S subunits are marked. Peaks corresponding to fractions 6 and 9 contain 30S and 50S subunits (see Supplementary material: Fig. Sl ). (b) Emission spectra (400–550 nm) of fluorescent mant-GDP and mant-GDPNP excited at 355 nm are shown with and without YqeH, as displayed in the inset. The fluorescence intensity is shown in arbitrary units (a.u.). (C–K) Fractions collected following co-sedimentation experiments with GST, GST-YlqF and GST-YqeH or its derivatives, were probed using anti-GST antibody in a Western blot. Shown on the right side of the gels (C–K) are the proteins used, indicating the domains they possess. (C) Purified GST was used as a negative control, as all constructs carry an N-terminal GST tag. (D) YlqF, which interacts with 50S, was used as a marker to identify the 50S fractions and as a positive control. Deletion constructs (H) ΔC-YqeH (residues 1–224) and (I) ΔN-YqeH (residues 64–366) and the stand-alone Zn finger (residues 1–46) domain (J) and PNR (residues 225–366) domain (K) are also indicated. The fractions corresponding to the ribosome profile are shown on the top and the nucleotides (GDP/GTP/GDPNP) used are indicated on the left. A high stoichiometric ratio of nucleotide (1 mM) to protein (500 nM) was used to ensure the desired nucleotide bound state.
    Figure Legend Snippet: YqeH binds 30S ribosomal subunit in the presence of GDPNP/GTP. (A) Co-sedimentation experiments were carried out by incubating purified ribosomes with GST-YqeH (or GST-YlqF) and nucleotides. Following centrifugation, the absorbance at 254 nm was monitored for the fractions collected (see Supplementary material for details). A representative ribosome profile thus obtained is shown here and peaks corresponding to 30S and 50S subunits are marked. Peaks corresponding to fractions 6 and 9 contain 30S and 50S subunits (see Supplementary material: Fig. Sl ). (b) Emission spectra (400–550 nm) of fluorescent mant-GDP and mant-GDPNP excited at 355 nm are shown with and without YqeH, as displayed in the inset. The fluorescence intensity is shown in arbitrary units (a.u.). (C–K) Fractions collected following co-sedimentation experiments with GST, GST-YlqF and GST-YqeH or its derivatives, were probed using anti-GST antibody in a Western blot. Shown on the right side of the gels (C–K) are the proteins used, indicating the domains they possess. (C) Purified GST was used as a negative control, as all constructs carry an N-terminal GST tag. (D) YlqF, which interacts with 50S, was used as a marker to identify the 50S fractions and as a positive control. Deletion constructs (H) ΔC-YqeH (residues 1–224) and (I) ΔN-YqeH (residues 64–366) and the stand-alone Zn finger (residues 1–46) domain (J) and PNR (residues 225–366) domain (K) are also indicated. The fractions corresponding to the ribosome profile are shown on the top and the nucleotides (GDP/GTP/GDPNP) used are indicated on the left. A high stoichiometric ratio of nucleotide (1 mM) to protein (500 nM) was used to ensure the desired nucleotide bound state.

    Techniques Used: Sedimentation, Purification, Centrifugation, Fluorescence, Western Blot, Negative Control, Construct, Marker, Positive Control

    12) Product Images from "A SelB/EF-Tu/aIF2γ-like protein from Methanosarcina mazei in the GTP-bound form binds cysteinyl-tRNACys"

    Article Title: A SelB/EF-Tu/aIF2γ-like protein from Methanosarcina mazei in the GTP-bound form binds cysteinyl-tRNACys

    Journal: Journal of Structural and Functional Genomics

    doi: 10.1007/s10969-015-9193-6

    Close-up stereo views of the switch I and II regions in EF-Tu ( a ) and MM1309 ( b ). The bound GMPPNP molecule and the Mg 2+ ion, and the EF-Tu and MM1309 residues in the switch I and II regions, which are involved in the GMPPNP interactions, are shown as stick models. The EF-Tu and MM1309 residues that are involved in the domain–domain interactions are also shown as stick models. The switch I and II regions of MM1309 are involved in domain–domain interactions, rather than GTP/GDP interactions. The switch I and II regions are colored pink and green , respectively. Transparent ribbon models of EF-Tu ( blue ) and MM1309 ( white ) are visible in the background
    Figure Legend Snippet: Close-up stereo views of the switch I and II regions in EF-Tu ( a ) and MM1309 ( b ). The bound GMPPNP molecule and the Mg 2+ ion, and the EF-Tu and MM1309 residues in the switch I and II regions, which are involved in the GMPPNP interactions, are shown as stick models. The EF-Tu and MM1309 residues that are involved in the domain–domain interactions are also shown as stick models. The switch I and II regions of MM1309 are involved in domain–domain interactions, rather than GTP/GDP interactions. The switch I and II regions are colored pink and green , respectively. Transparent ribbon models of EF-Tu ( blue ) and MM1309 ( white ) are visible in the background

    Techniques Used:

    ITC analysis. The upper and lower panels display the ITC titration curves and the binding isotherms, respectively, for MM1309 with GTP·Mg 2+ ( a ), GTP without Mg 2+ ( b ), GDP·Mg 2+ ( c ), and GMPPNP·Mg 2+ ( d ). N , the binding stoichiometry; K b , the observed binding constant; K d ( K d = 1/ K b ), the dissociation constant; ∆ H , the binding enthalpy; ∆ S , the binding entropy
    Figure Legend Snippet: ITC analysis. The upper and lower panels display the ITC titration curves and the binding isotherms, respectively, for MM1309 with GTP·Mg 2+ ( a ), GTP without Mg 2+ ( b ), GDP·Mg 2+ ( c ), and GMPPNP·Mg 2+ ( d ). N , the binding stoichiometry; K b , the observed binding constant; K d ( K d = 1/ K b ), the dissociation constant; ∆ H , the binding enthalpy; ∆ S , the binding entropy

    Techniques Used: Titration, Binding Assay

    Stereo views of the GTP binding sites. a The bound GMPPNP molecule in the T. aquaticus EF-Tu·GMPPNP·Mg 2+ structure. b The bound GMPPNP molecule in the MM1309·GMPPNP·Mg 2+ structure. The F o – F c omit map (contoured at 3.3 σ) of the bound GMPPNP·Mg 2+ in the MM1309 active site. c , d Close-up stereo views around the γ-phosphate group of the bound GMPPNP in T. aquaticus EF-Tu·GMPPNP·Mg 2+ ( c ) and MM1309·GMPPNP·Mg 2+ ( d ). The amino acid residues surrounding the phosphate groups and the magnesium ions of the bound GMPPNP·Mg 2+ are depicted by stick models. e The bound GDP molecule in the MM1309·GDP structure. The F o – F c omit map (contoured at 4.0 σ) of the bound GDP·Mg 2+ in the MM1309 active site. f The GTP binding site in the MM1309 apo form. The MM1309 residues that are located close to the bound guanine nucleotide are represented as stick models. The P-loop motifs (Gly17–Thr25 in EF-Tu and Gly7–Thr15 in MM1309) are shown in sky blue . The switch I regions are colored pink . Transparent ribbon models of EF-Tu ( blue ) and MM1309 ( white ) are visible in the background
    Figure Legend Snippet: Stereo views of the GTP binding sites. a The bound GMPPNP molecule in the T. aquaticus EF-Tu·GMPPNP·Mg 2+ structure. b The bound GMPPNP molecule in the MM1309·GMPPNP·Mg 2+ structure. The F o – F c omit map (contoured at 3.3 σ) of the bound GMPPNP·Mg 2+ in the MM1309 active site. c , d Close-up stereo views around the γ-phosphate group of the bound GMPPNP in T. aquaticus EF-Tu·GMPPNP·Mg 2+ ( c ) and MM1309·GMPPNP·Mg 2+ ( d ). The amino acid residues surrounding the phosphate groups and the magnesium ions of the bound GMPPNP·Mg 2+ are depicted by stick models. e The bound GDP molecule in the MM1309·GDP structure. The F o – F c omit map (contoured at 4.0 σ) of the bound GDP·Mg 2+ in the MM1309 active site. f The GTP binding site in the MM1309 apo form. The MM1309 residues that are located close to the bound guanine nucleotide are represented as stick models. The P-loop motifs (Gly17–Thr25 in EF-Tu and Gly7–Thr15 in MM1309) are shown in sky blue . The switch I regions are colored pink . Transparent ribbon models of EF-Tu ( blue ) and MM1309 ( white ) are visible in the background

    Techniques Used: Binding Assay

    Superposition of MM1309 with EF-Tu, aSelB, and aIF2γ, represented by ribbon models. a Superposition of the MM1309 structures in the GMPPNP-bound, GDP-bound, and apo forms. b Superposition of MM1309 with T. aquaticus EF-Tu in the GTP-bound form (PDB code: 1TTT). c Superposition of MM1309 with T. aquaticus EF-Tu in the GDP-bound form (PDB code: 1TUI). d Superposition of MM1309 with M. maripaludis aSelB (PDB code: 4ACA) and with P. abyssi aIF2γ (PDB code: 1KK0)
    Figure Legend Snippet: Superposition of MM1309 with EF-Tu, aSelB, and aIF2γ, represented by ribbon models. a Superposition of the MM1309 structures in the GMPPNP-bound, GDP-bound, and apo forms. b Superposition of MM1309 with T. aquaticus EF-Tu in the GTP-bound form (PDB code: 1TTT). c Superposition of MM1309 with T. aquaticus EF-Tu in the GDP-bound form (PDB code: 1TUI). d Superposition of MM1309 with M. maripaludis aSelB (PDB code: 4ACA) and with P. abyssi aIF2γ (PDB code: 1KK0)

    Techniques Used:

    13) Product Images from "The GTPase Activity of Murine Guanylate-binding Protein 2 (mGBP2) Controls the Intracellular Localization and Recruitment to the Parasitophorous Vacuole of Toxoplasma gondii *"

    Article Title: The GTPase Activity of Murine Guanylate-binding Protein 2 (mGBP2) Controls the Intracellular Localization and Recruitment to the Parasitophorous Vacuole of Toxoplasma gondii *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M112.379636

    Nucleotide-dependent multimerization. Size-exclusion chromatography of WT ( A ) and mutant mGBP2 R48A ( B ), K51A ( C ), E99A ( D ), and D182N ( E ) bound to GTPγS, GDP, GMP, and in the nucleotide-free state at 4 °C. Elution of all proteins was
    Figure Legend Snippet: Nucleotide-dependent multimerization. Size-exclusion chromatography of WT ( A ) and mutant mGBP2 R48A ( B ), K51A ( C ), E99A ( D ), and D182N ( E ) bound to GTPγS, GDP, GMP, and in the nucleotide-free state at 4 °C. Elution of all proteins was

    Techniques Used: Size-exclusion Chromatography, Mutagenesis

    Related Articles

    Recombinant:

    Article Title: The GTPase Activity of Murine Guanylate-binding Protein 2 (mGBP2) Controls the Intracellular Localization and Recruitment to the Parasitophorous Vacuole of Toxoplasma gondii *
    Article Snippet: .. Therefore, the nucleotide binding affinities of recombinant WT and mutant proteins were determined using fluorescence spectroscopy with guanine nucleotides labeled with the fluorescent mant group. shows protein titration curves for mant-GTPγS ( B ), a nonhydrolyzable analog of GTP, mant-GDP ( C ), and mant-GMP ( D ). .. Binding of mant-nucleotides to the WT protein resulted in a dose-dependent increase of fluorescence.

    Fluorescence:

    Article Title: The GTPase Activity of Murine Guanylate-binding Protein 2 (mGBP2) Controls the Intracellular Localization and Recruitment to the Parasitophorous Vacuole of Toxoplasma gondii *
    Article Snippet: .. Therefore, the nucleotide binding affinities of recombinant WT and mutant proteins were determined using fluorescence spectroscopy with guanine nucleotides labeled with the fluorescent mant group. shows protein titration curves for mant-GTPγS ( B ), a nonhydrolyzable analog of GTP, mant-GDP ( C ), and mant-GMP ( D ). .. Binding of mant-nucleotides to the WT protein resulted in a dose-dependent increase of fluorescence.

    Mutagenesis:

    Article Title: The GTPase Activity of Murine Guanylate-binding Protein 2 (mGBP2) Controls the Intracellular Localization and Recruitment to the Parasitophorous Vacuole of Toxoplasma gondii *
    Article Snippet: .. Therefore, the nucleotide binding affinities of recombinant WT and mutant proteins were determined using fluorescence spectroscopy with guanine nucleotides labeled with the fluorescent mant group. shows protein titration curves for mant-GTPγS ( B ), a nonhydrolyzable analog of GTP, mant-GDP ( C ), and mant-GMP ( D ). .. Binding of mant-nucleotides to the WT protein resulted in a dose-dependent increase of fluorescence.

    Labeling:

    Article Title: The GTPase Activity of Murine Guanylate-binding Protein 2 (mGBP2) Controls the Intracellular Localization and Recruitment to the Parasitophorous Vacuole of Toxoplasma gondii *
    Article Snippet: .. Therefore, the nucleotide binding affinities of recombinant WT and mutant proteins were determined using fluorescence spectroscopy with guanine nucleotides labeled with the fluorescent mant group. shows protein titration curves for mant-GTPγS ( B ), a nonhydrolyzable analog of GTP, mant-GDP ( C ), and mant-GMP ( D ). .. Binding of mant-nucleotides to the WT protein resulted in a dose-dependent increase of fluorescence.

    Titration:

    Article Title: The GTPase Activity of Murine Guanylate-binding Protein 2 (mGBP2) Controls the Intracellular Localization and Recruitment to the Parasitophorous Vacuole of Toxoplasma gondii *
    Article Snippet: .. Therefore, the nucleotide binding affinities of recombinant WT and mutant proteins were determined using fluorescence spectroscopy with guanine nucleotides labeled with the fluorescent mant group. shows protein titration curves for mant-GTPγS ( B ), a nonhydrolyzable analog of GTP, mant-GDP ( C ), and mant-GMP ( D ). .. Binding of mant-nucleotides to the WT protein resulted in a dose-dependent increase of fluorescence.

    other:

    Article Title: The GTPase Activity of Murine Guanylate-binding Protein 2 (mGBP2) Controls the Intracellular Localization and Recruitment to the Parasitophorous Vacuole of Toxoplasma gondii *
    Article Snippet: WT mGBP2 showed comparable affinities to mant-GTPγS and mant-GDP with Kd values of 0.45 and 0.54 μ m , respectively, whereas the affinity to mant-GMP was 30-fold weaker ( Kd = 14.4 μ m ) ( ).

    Article Title: Role and timing of GTP binding and hydrolysis during EF-G-dependent tRNA translocation on the ribosome
    Article Snippet: Mant-labeled GDP, GTP, and GDPNP were purchased from Jena Bioscience (Jena, Germany).

    Spectroscopy:

    Article Title: The GTPase Activity of Murine Guanylate-binding Protein 2 (mGBP2) Controls the Intracellular Localization and Recruitment to the Parasitophorous Vacuole of Toxoplasma gondii *
    Article Snippet: .. Therefore, the nucleotide binding affinities of recombinant WT and mutant proteins were determined using fluorescence spectroscopy with guanine nucleotides labeled with the fluorescent mant group. shows protein titration curves for mant-GTPγS ( B ), a nonhydrolyzable analog of GTP, mant-GDP ( C ), and mant-GMP ( D ). .. Binding of mant-nucleotides to the WT protein resulted in a dose-dependent increase of fluorescence.

    Binding Assay:

    Article Title: Circularly permuted GTPase YqeH binds 30S ribosomal subunit: Implications for its role in ribosome assembly
    Article Snippet: .. The nucleotide binding experiments were carried out with 5 μM YqeH (or GST) and 200 nM mant-GDP or mant-GDPNP (Jena Biosciences) as detailed in . .. After incubation, the fluorescent nucleotides were excited at 355 nm and emission (at 448 nm) was monitored between 400 and 600 nm using a spectrofluorimeter (Perkin-Elmer).

    Article Title: The GTPase Activity of Murine Guanylate-binding Protein 2 (mGBP2) Controls the Intracellular Localization and Recruitment to the Parasitophorous Vacuole of Toxoplasma gondii *
    Article Snippet: .. Therefore, the nucleotide binding affinities of recombinant WT and mutant proteins were determined using fluorescence spectroscopy with guanine nucleotides labeled with the fluorescent mant group. shows protein titration curves for mant-GTPγS ( B ), a nonhydrolyzable analog of GTP, mant-GDP ( C ), and mant-GMP ( D ). .. Binding of mant-nucleotides to the WT protein resulted in a dose-dependent increase of fluorescence.

    Article Title: The GTPase Activity of Murine Guanylate-binding Protein 2 (mGBP2) Controls the Intracellular Localization and Recruitment to the Parasitophorous Vacuole of Toxoplasma gondii *
    Article Snippet: .. This is remarkable as the binding affinities to mant-GTPγS and mant-GDP were comparable with the WT , and the affinity to mant-Gpp(NH)p was even slightly stronger ( and ). .. In the case of the K51A mutant, no dimer formation was observed independently of the offered nucleotide ( C , , and ).

    Article Title: Circularly permuted GTPase YqeH binds 30S ribosomal subunit: Implications for its role in ribosome assembly
    Article Snippet: .. To ascertain that the nucleotides do bind the protein, fluorescent nucleotide binding to YqeH was examined using mant-GDP, mant-GDPNP. .. An increased fluorescence of the mant nucleotides in presence of YqeH suggests their binding to the protein ( B).

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    Jena Bioscience mant gdp
    Replacement of <t>mant-GMPPNP</t> by <t>mant-GDP</t> eliminates the dissociation of Rac1 · RhoGDI complexes by anionic liposomes and TrioN, but nucleotide exchange on Rac1 in complex with RhoGDI is conserved. 2 nmol of Rac1(GDP)·RhoGDI complex and 10
    Mant Gdp, supplied by Jena Bioscience, used in various techniques. Bioz Stars score: 94/100, based on 23 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Replacement of mant-GMPPNP by mant-GDP eliminates the dissociation of Rac1 · RhoGDI complexes by anionic liposomes and TrioN, but nucleotide exchange on Rac1 in complex with RhoGDI is conserved. 2 nmol of Rac1(GDP)·RhoGDI complex and 10

    Journal:

    Article Title: Dissociation of Rac1(GDP)?RhoGDI Complexes by the Cooperative Action of Anionic Liposomes Containing Phosphatidylinositol 3,4,5-Trisphosphate, Rac Guanine Nucleotide Exchange Factor, and GTP *

    doi: 10.1074/jbc.M800734200

    Figure Lengend Snippet: Replacement of mant-GMPPNP by mant-GDP eliminates the dissociation of Rac1 · RhoGDI complexes by anionic liposomes and TrioN, but nucleotide exchange on Rac1 in complex with RhoGDI is conserved. 2 nmol of Rac1(GDP)·RhoGDI complex and 10

    Article Snippet: The fluorescent hydrolysis-resistant GTP analogues mant-GMPPNP and mant-GDP were obtained from Jena Bioscience GmbH.

    Techniques:

    Exposure of Rac1(GDP) · RhoGDI complexes to anionic liposomes, mant-GMMPNP, and TrioN causes dissociation of complexes, GDP to mant-GMPPNP exchange on Rac1, and binding of Rac1-GMPPNP to liposomes. 2 nmol of Rac1(GDP)·RhoGDI complex were

    Journal:

    Article Title: Dissociation of Rac1(GDP)?RhoGDI Complexes by the Cooperative Action of Anionic Liposomes Containing Phosphatidylinositol 3,4,5-Trisphosphate, Rac Guanine Nucleotide Exchange Factor, and GTP *

    doi: 10.1074/jbc.M800734200

    Figure Lengend Snippet: Exposure of Rac1(GDP) · RhoGDI complexes to anionic liposomes, mant-GMMPNP, and TrioN causes dissociation of complexes, GDP to mant-GMPPNP exchange on Rac1, and binding of Rac1-GMPPNP to liposomes. 2 nmol of Rac1(GDP)·RhoGDI complex were

    Article Snippet: The fluorescent hydrolysis-resistant GTP analogues mant-GMPPNP and mant-GDP were obtained from Jena Bioscience GmbH.

    Techniques: Binding Assay

    Nucleotide-dependent multimerization. Size-exclusion chromatography of WT ( A ) and mutant mGBP2 R48A ( B ), K51A ( C ), E99A ( D ), and D182N ( E ) bound to GTPγS, GDP, GMP, and in the nucleotide-free state at 4 °C. Elution of all proteins was

    Journal: The Journal of Biological Chemistry

    Article Title: The GTPase Activity of Murine Guanylate-binding Protein 2 (mGBP2) Controls the Intracellular Localization and Recruitment to the Parasitophorous Vacuole of Toxoplasma gondii *

    doi: 10.1074/jbc.M112.379636

    Figure Lengend Snippet: Nucleotide-dependent multimerization. Size-exclusion chromatography of WT ( A ) and mutant mGBP2 R48A ( B ), K51A ( C ), E99A ( D ), and D182N ( E ) bound to GTPγS, GDP, GMP, and in the nucleotide-free state at 4 °C. Elution of all proteins was

    Article Snippet: This is remarkable as the binding affinities to mant-GTPγS and mant-GDP were comparable with the WT , and the affinity to mant-Gpp(NH)p was even slightly stronger ( and ).

    Techniques: Size-exclusion Chromatography, Mutagenesis

    YqeH binds 30S ribosomal subunit in the presence of GDPNP/GTP. (A) Co-sedimentation experiments were carried out by incubating purified ribosomes with GST-YqeH (or GST-YlqF) and nucleotides. Following centrifugation, the absorbance at 254 nm was monitored for the fractions collected (see Supplementary material for details). A representative ribosome profile thus obtained is shown here and peaks corresponding to 30S and 50S subunits are marked. Peaks corresponding to fractions 6 and 9 contain 30S and 50S subunits (see Supplementary material: Fig. Sl ). (b) Emission spectra (400–550 nm) of fluorescent mant-GDP and mant-GDPNP excited at 355 nm are shown with and without YqeH, as displayed in the inset. The fluorescence intensity is shown in arbitrary units (a.u.). (C–K) Fractions collected following co-sedimentation experiments with GST, GST-YlqF and GST-YqeH or its derivatives, were probed using anti-GST antibody in a Western blot. Shown on the right side of the gels (C–K) are the proteins used, indicating the domains they possess. (C) Purified GST was used as a negative control, as all constructs carry an N-terminal GST tag. (D) YlqF, which interacts with 50S, was used as a marker to identify the 50S fractions and as a positive control. Deletion constructs (H) ΔC-YqeH (residues 1–224) and (I) ΔN-YqeH (residues 64–366) and the stand-alone Zn finger (residues 1–46) domain (J) and PNR (residues 225–366) domain (K) are also indicated. The fractions corresponding to the ribosome profile are shown on the top and the nucleotides (GDP/GTP/GDPNP) used are indicated on the left. A high stoichiometric ratio of nucleotide (1 mM) to protein (500 nM) was used to ensure the desired nucleotide bound state.

    Journal: Biochemical and Biophysical Research Communications

    Article Title: Circularly permuted GTPase YqeH binds 30S ribosomal subunit: Implications for its role in ribosome assembly

    doi: 10.1016/j.bbrc.2009.06.078

    Figure Lengend Snippet: YqeH binds 30S ribosomal subunit in the presence of GDPNP/GTP. (A) Co-sedimentation experiments were carried out by incubating purified ribosomes with GST-YqeH (or GST-YlqF) and nucleotides. Following centrifugation, the absorbance at 254 nm was monitored for the fractions collected (see Supplementary material for details). A representative ribosome profile thus obtained is shown here and peaks corresponding to 30S and 50S subunits are marked. Peaks corresponding to fractions 6 and 9 contain 30S and 50S subunits (see Supplementary material: Fig. Sl ). (b) Emission spectra (400–550 nm) of fluorescent mant-GDP and mant-GDPNP excited at 355 nm are shown with and without YqeH, as displayed in the inset. The fluorescence intensity is shown in arbitrary units (a.u.). (C–K) Fractions collected following co-sedimentation experiments with GST, GST-YlqF and GST-YqeH or its derivatives, were probed using anti-GST antibody in a Western blot. Shown on the right side of the gels (C–K) are the proteins used, indicating the domains they possess. (C) Purified GST was used as a negative control, as all constructs carry an N-terminal GST tag. (D) YlqF, which interacts with 50S, was used as a marker to identify the 50S fractions and as a positive control. Deletion constructs (H) ΔC-YqeH (residues 1–224) and (I) ΔN-YqeH (residues 64–366) and the stand-alone Zn finger (residues 1–46) domain (J) and PNR (residues 225–366) domain (K) are also indicated. The fractions corresponding to the ribosome profile are shown on the top and the nucleotides (GDP/GTP/GDPNP) used are indicated on the left. A high stoichiometric ratio of nucleotide (1 mM) to protein (500 nM) was used to ensure the desired nucleotide bound state.

    Article Snippet: To ascertain that the nucleotides do bind the protein, fluorescent nucleotide binding to YqeH was examined using mant-GDP, mant-GDPNP.

    Techniques: Sedimentation, Purification, Centrifugation, Fluorescence, Western Blot, Negative Control, Construct, Marker, Positive Control

    YqeH binds a non-specific RNA. EMSA was carried out with YqeH or the indicated domains, in presence of either ssRNA or dsRNA. The migration of ssRNA and dsRNA is shown as control in the lanes at the extreme left and is indicated by an arrow. A retarded migration of RNA in presence of YqeH is indicated by ’shift’. The nucleotide bound states of YqeH are indicated on the top. (A) Both ssRNA and dsRNA are retarded in the presence of YqeH. The dissociation of dsRNA into ssRNAs is not observed in nucleotide-free, GDP and GTP-bound forms. The presence of S5 is indicated above the lanes. The apparent reduction in the intensity of free dsRNAs in the presence of S5 when compared to the corresponding lanes containing YqeH alone suggests a potential increase in YqeH–RNA interactions (the last three lanes in the right). (B) Like in (A), the migration of ssRNA and the mixture of complementary ssRNAs is shown in the lanes at the extreme left as controls and is indicated by an arrow. Increasing concentration of YqeH (2.5, 5, 10 μM) is depicted by a triangle on the top. No annealing activity for YqeH was apparent. (C) EMSA carried out with GST, N (NTD) and C (CTD) terminal domains of YqeH (indicated above the lanes) showed no apparent shift in migration of ssRNA and dsRNA. (D) Also in the presence of deletion constructs (ΔN and ΔC-YqeH), no shift in dsRNA migration was observed.

    Journal: Biochemical and Biophysical Research Communications

    Article Title: Circularly permuted GTPase YqeH binds 30S ribosomal subunit: Implications for its role in ribosome assembly

    doi: 10.1016/j.bbrc.2009.06.078

    Figure Lengend Snippet: YqeH binds a non-specific RNA. EMSA was carried out with YqeH or the indicated domains, in presence of either ssRNA or dsRNA. The migration of ssRNA and dsRNA is shown as control in the lanes at the extreme left and is indicated by an arrow. A retarded migration of RNA in presence of YqeH is indicated by ’shift’. The nucleotide bound states of YqeH are indicated on the top. (A) Both ssRNA and dsRNA are retarded in the presence of YqeH. The dissociation of dsRNA into ssRNAs is not observed in nucleotide-free, GDP and GTP-bound forms. The presence of S5 is indicated above the lanes. The apparent reduction in the intensity of free dsRNAs in the presence of S5 when compared to the corresponding lanes containing YqeH alone suggests a potential increase in YqeH–RNA interactions (the last three lanes in the right). (B) Like in (A), the migration of ssRNA and the mixture of complementary ssRNAs is shown in the lanes at the extreme left as controls and is indicated by an arrow. Increasing concentration of YqeH (2.5, 5, 10 μM) is depicted by a triangle on the top. No annealing activity for YqeH was apparent. (C) EMSA carried out with GST, N (NTD) and C (CTD) terminal domains of YqeH (indicated above the lanes) showed no apparent shift in migration of ssRNA and dsRNA. (D) Also in the presence of deletion constructs (ΔN and ΔC-YqeH), no shift in dsRNA migration was observed.

    Article Snippet: The nucleotide binding experiments were carried out with 5 μM YqeH (or GST) and 200 nM mant-GDP or mant-GDPNP (Jena Biosciences) as detailed in .

    Techniques: Migration, Concentration Assay, Activity Assay, Construct